Therapeutic efficacy of many biologically active substances has been hindered due to the difficulty or incapability to deliver the substance in therapeutically active, or sufficient amounts to the patient's organ or tissue to be treated. One of the main difficulties with respect to the efficient delivery of such substances is presented by biological barriers with low or no permeability to many substances.
Compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both polar and non-polar components within the body, and therefore, should typically contain themselves both polar and non-polar domains. For this purpose, amphiphilic compounds containing polar and hydrophobic (non-polar) domains and, particularly, cationic amphiphiles have been found suitable for intracellular delivery of drugs.
Vesicles from phospholipid amphiphiles known as liposomes are used to deliver drugs for treating certain cancers and microbial infections. For example, the therapeutic efficacy of some important chemotherapeutic and antibiotic agents can be enhanced by encapsulating them in liposomes, which have an acceptable blood circulatory lifetime and improved access to diseased sites via increased vascular porosity (Lasic, 1996). However the mode of delivery in these cases is passive, and the goal of targeting with controlled release still remains elusive.
Targeted controlled release of biologically active materials for therapeutic applications is under continuous development using different drug delivery platforms such as micelles, emulsions, complexants, prodrugs and vesicles. Vesicles, made from synthetic amphiphiles, as well as liposomes, made from synthetic or natural phospholipids, are considered very promising approaches because the therapeutic agent is totally isolated from the environment, each vesicle or liposome delivers many molecules, and the surface properties of the vesicle or liposome can be modified for biological stability, enhanced penetration through biological barriers and targeting, independent of the physico-chemical properties of the encapsulated drug. However, in spite of the serious efforts invested in making vesicles with targeted controlled release properties, vesicles with good mechanical, chemical and biological stability needed for many applications have not yet been produced.
Three approaches are currently being pursued to improve temporal control of drug release from vesicles or liposomes: (i) physical approaches such as temperature, pH or target-binding sensitive-induced phase changes; (ii) chemical approaches, which try to destabilize liposomes at a particular location; and (iii) biological approaches, which use surface attached targeting moieties (Lasic, 1998).
Spatial control of drug release has been attempted by cellular targeting in physically accessible sites by liposomes containing antibody molecules attached to liposome surface by polymer chains. The state of the art is ambivalent about the potential of antibodies and surface ligands for targeted delivery. It seems that problems of accessibility to a particular tissue and cells as well as overlooked severity of triggered immune response to the host organism by antibody- or lectin-coated liposomes, makes this approach problematic.
Enzyme-labile liposomes have been investigated as a model for site-directed liposomal drug delivery system. This approach takes into account that certain pathological cells produce excessive amounts of a particular enzyme, for example, bone cancer cells produce alkaline phosphatase, and neuroblastoma cells produce acetylcholinesterase. In one approach, small unilamellar vesicles with acetylcholine headgroups were made and shown to disrupt in vitro in the presence of acetylcholinesterase (Menger and Johnston, 1991).
Elastase is of interest because of its ubiquitous involvement in inflammatory and tumorigenic conditions. A peptide-lipid conjugate sensitive to enzymatic cleavage was designed and reported to generate liposomes that could be triggered to fuse by enzymatic activation. In effect, covalent linkage of dioleoyl phosphatidylethanolamine to an elastase substrate, N-acetyl-ala-ala, resulted in a cleavable peptide-lipid with no intrinsic fusogenic activity. In addition, the ability of elastase to recognize a simple peptide substrate simplifies coupling as well as potentially limiting the immunogenicity of the peptide-lipid (Pak et al., 1998).
U.S. Pat. No. 6,087,325 describes peptide-lipid conjugates that are incorporated into liposomes so as to selectively destabilize the liposomes in the vicinity of target peptidase-secreting cells, and hence to deliver the liposomes to the vicinity of the target cells, or directly into the cells. The liposomes can thus be used to treat mammals for diseases, disorders or conditions such as tumors, microbial infection and inflammation characterized by the occurrence of peptidase-secreting cells.
Vesicles and liposomes may also be destabilized by changes in pH at the target site. For example, WO 01/05375 describes amphiphilic lipid compounds having an acid or oxidative labile vinyl ether linked hydrophilic headgroup and liposomes made therewith, of a given stability for circulation and of a desired release rate profile or fusogenicity to suit a particular therapeutic or diagnostic indication. The stabilized liposomes or vesicles are said to be readily destabilized at a given site because of changes in the chemical environment such as pH or oxidants.
U.S. Pat. No. 6,294,191 describes liposomes containing one or more N-acylated phosphatidylethanolamine moieties for localizing the delivery of bioactive agents to cells.
The transport of compounds from the blood to target tissues is restricted by biological barriers such as the blood-brain-barrier (BBB). Drug delivery to the brain is particularly hampered because of the tight junctions between adjacent endothelial cells of brain capillaries, which form the BBB. However, some lipid soluble substances can penetrate passively across this barrier, whereas hydrophilic and ionic substances [e.g., amino acids] are transported by a specific carrier transport system. Therefore, it is possible to improve the entry of certain substances into the central nervous system (CNS) by regulating these transporters.
Efforts have been made to enhance transport via the BBB by conjugating drugs with CNS permeable moieties. For example, attempts have been made in correcting disorders affecting the CNS system by increasing BBB permeability of exogenous biological compounds such as proteins or specific nucleic acid sequences by conjugating them with lipids (Chopineau et al., 1998). Long alkyl chains, such as fatty acids, due to their hydrophobicity and low toxicity, are good potential candidates that may enhance BBB permeability of drugs when covalently bound to them. Another example of forming conjugates with a vector or carrier that enhance transport across the BBB is the utilization of membrane-bound enzymes to release a bioactive peptide from a highly lipophilic triglyceride peptide carrier (Patel et al., 1997).
The methods above and others, however, do not encapsulate the active agent and are not selective as the conjugated molecule is adsorbed and degraded in several biological compartments. Some further studies have been reported on the use of liposomes for crossing the BBB such as in corporation of enzymes into the liposomes (Naoi and Yagi et. al, 1980); using PC cholesterol and sulfatides, for delivery of their contents to rat brains (Yagi et. al, 1982); using liposomes composed of PC cholesterol and p-aminophenyl-alpha-mannopyranoside to deliver [3H]-galactocerebroside to brain lysosomes and to glial cells because of recognition of mannose residues by the cells of the BBB (Umezawa and Eto, 1988); and a study in which cAMP phosphodiesterase [PDE] was iodinated, entrapped in either dehydration-rehydration vesicles or small unilamellar vesicles (SUV's) prepared from PC cholesterol and sulfatides, and delivered to the brain by means of intravenous injection with hyperosmolar mannitol, that was shown to increase the permeability of the liposomes through the BBB (Kozler, 2001).
None of the above approaches of the prior art provide effective targeting to the CNS. In addition, because of poor stability, state of art liposomes release a relatively large amount of the encapsulated active agent into a variety of non-targeted tissues.